Lavan B E, Kuhné M R, Garner C W, Anderson D, Reedijk M, Pawson T, Lienhard G E
Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03755-3844.
J Biol Chem. 1992 Jun 5;267(16):11631-6.
The interactions of the phosphotyrosine (Tyr(P))-containing proteins in basal and insulin-stimulated 3T3-L1 adipocytes with src homology 2 (SH2) domains from phosphatidylinositol 3-kinase (PI3K), ras GTPase-activating protein (GAP), and phospholipase C gamma have been examined. The Tyr(P) forms of the insulin receptor and its 160-kDa substrate protein (pp160) associated with fusion proteins containing either or both the SH2 domains of PI3K, but not with fusion proteins containing the two SH2 domains of GAP or phospholipase C gamma. These results demonstrate a specificity for the association of the Tyr(P) form of the insulin receptor and pp160 with SH2 domains that parallels the reported effects of insulin on PI3K, GAP, and phospholipase C gamma in vivo. Immunoprecipitates of pp160 from the cytosol of insulin-treated, but not basal, 3T3-L1 adipocytes contained PI3K activity. Moreover, the Tyr(P) form of pp160 with associated PI3K activity migrated at 10 S on a sucrose velocity gradient, whereas the Tyr(P) form without associated activity migrated at 6 S. These findings indicate that the Tyr(P) form of pp160 associates directly with PI3K in vivo.
已对基础状态和胰岛素刺激的3T3-L1脂肪细胞中含磷酸酪氨酸（Tyr(P)）的蛋白质与磷脂酰肌醇3激酶（PI3K）、ras GTP酶激活蛋白（GAP）和磷脂酶Cγ的src同源2（SH2）结构域之间的相互作用进行了研究。胰岛素受体及其160 kDa底物蛋白（pp160）的Tyr(P)形式与含有PI3K的一个或两个SH2结构域的融合蛋白相关，但与含有GAP或磷脂酶Cγ的两个SH2结构域的融合蛋白不相关。这些结果证明了胰岛素受体和pp160的Tyr(P)形式与SH2结构域结合具有特异性，这与胰岛素在体内对PI3K、GAP和磷脂酶Cγ的报道作用相似。从胰岛素处理而非基础状态的3T3-L1脂肪细胞的胞质溶胶中免疫沉淀的pp160含有PI3K活性。此外，具有相关PI3K活性的pp160的Tyr(P)形式在蔗糖速度梯度上以10 S迁移，而没有相关活性的Tyr(P)形式以6 S迁移。这些发现表明pp160的Tyr(P)形式在体内直接与PI3K结合。
